Methods of treating and preventing diseases and disorders of the central nervous system

ABSTRACT

Disclosed is a method of treating or preventing a disease or disorder of the central nervous system (CNS) in a patient comprising administering transcranially, for example, directly to the skull, an effective amount of an anti-inflammatory agent to the patient. Examples of the anti-inflammatory agent include glutathione and inhibitors of purinergic receptors such as P2X 4 , P2X 7 , P2Y 6 , and P2Y 12  receptors. Examples of disease or disorder of the CNS include brain injury, particularly traumatic brain injury, inflammation, infection, degeneration of brain cells, stroke, brain edema, tumor, Alzheimer&#39;s disease, Parkinson&#39;s disease, and multiple sclerosis. Also disclosed is a kit comprising at least one anti-inflammatory agent and printed materials containing instructions for transcranially administering the anti-inflammatory agent to the patient having a disease or disorder of the CNS.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/599,107, filed Feb. 15, 2012, the disclosure of whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

The central nervous system (CNS) of an animal, for example, human,contains several important receptor systems such as the glutamate,acetylcholine, GABA_(A), and NMDA receptor systems, and injury to theCNS as well as other diseases and disorders of the CNS could be quiteserious including threat to life. For example, following a traumaticinjury to the CNS, a cascade of physiological events can lead toneuronal loss, including, for example, an inflammatory immune responseand excitotoxicity resulting from the initial impact of one or more ofthe above receptor systems. In addition, traumatic CNS injury can beaccompanied by brain edema that enhances the cascade of injury and canlead to further secondary cell death and increased mortality of theanimal.

An example of the CNS injury is traumatic brain injury (TBI) to thebrain, which is a head injury caused by trauma to the brain. Symptoms ofTBI include headache, confusion, dizziness, blurred vision, changes inmood, impairment of cognitive function such as memory, learning, andattention, nausea, convulsions or seizures, slurring of speech, numbnessof extremities, and loss of coordination. While some symptoms appearimmediately, others do not appear until days, months or even years afterthe TBI event. TBI is a major cause of preventable death and morbidity,and disability following injury in war zones, in sports and recreation,and general population falls and accidents. Secondary injury occurringhours and days after the head trauma occurs as a result of ischemia,blood brain barrier leakage and inflammatory/oxidative stress.

Attempts to treat diseases and disorders of the CNS, particularly TBI,that target neuroprotection following TBI have proven to be of littleefficacy. The blood brain barrier (BBB) has been a cause of many drugs'ineffective translation from “promising” to “failure” in the treatmentor prevention of various brain injury and other CNS disease ordisorders. Much resources have been spent on developing drugs which whenadministered systemically have proven to be ineffective. This failure isbelieved to be due to the blockage of the drug by the BBB from reachingthe CNS, wherein the blockage leads to inadequate concentration of thedrug in the CNS.

The foregoing shows that there is an unmet need for a treatment modalitythat can effectively administer a drug to the CNS, thereby treatingand/or preventing a disease or disorder of the CNS.

BRIEF SUMMARY OF THE INVENTION

The foregoing need has been fulfilled by the invention. Accordingly, theinvention provides a method of treating or preventing a disease ordisorder of the central nervous system (CNS) in a patient comprisingadministering transcranially an effective amount of reactive oxygenscavenger (ROS) or an anti-inflammatory agent to the patient. Theinvention further provides a method of inhibiting, reducing, oreliminating the formation of reactive microglia in a patient sufferingfrom a traumatic brain injury comprising administering transcranially aneffective amount of an anti-inflammatory agent to the patient.

The invention also provides a method of inhibiting, reducing, oreliminating the recruitment of neutrophils and/or monocytes in a patientsuffering from a traumatic brain injury comprising administeringtranscranially an effective amount of an anti-inflammatory agent to thepatient. The invention further provides a method of reducing the numberof dead cells in the brain parenchyma or meninges in a patient sufferingfrom a traumatic brain injury comprising administering transcranially aneffective amount of an anti-inflammatory agent to the patient. Theinvention also provides a kit comprising at least one anti-inflammatoryagent and printed materials containing instructions for transcraniallyadministering the anti-inflammatory agent to the patient having adisease or disorder of the central nervous system (CNS).

The present invention provides methods wherein therapeutic agentspenetrate the skull and pass into the cerebrospinal fluid. The approachof the present invention allows the therapeutic agents bypass the bloodbrain barrier (BBB) and the agents rapidly reach the injured or inflamedareas of the CNS. The invention provides methods wherein a high localconcentration of the therapeutic agent is quickly established at thesite of the CNS injury.

In embodiments of the invention, the reactive oxygen scavengers rapidlyeliminate free radicals generated as a result of the CNS injury. Inembodiments of the invention, purinergic antagonists block the damagesensing ability of the purinergic receptors, thereby stopping the innateimmune cells to activate the brain resident microglia or prevent thearrival of peripherally-derived neutrophils, which are the two innateinflammatory cells. The invention also provides a pharmaceuticalcomposition for transcranially treating or preventing a disease ordisorder of the central nervous system (CNS) comprising ananti-inflammatory agent as the main ingredient. The pharmaceuticalcomposition is adapted for or is suitable for transcranialadministration.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 depicts the number of dead cells in the meninges and parenchymaof mice as a function of time after meningeal compression injury.

FIG. 2 illustrates the distribution of fluorescent probes in the brainas a function of their molecular weight after transcranialadministration of the probes to intact (non-thinned) skull of mice.White indicates that the probes were found in the subarachnoid space ofthe brain and black indicates they were not present therein. Grayindicates that the probe was not tested at that time.

FIG. 3 depicts the percentage of microglia with jellyfish morphologyfound after administration of purinergic receptor antagonists to theskull of mice prior to inducing meningeal compression injury. MRS2578(500 μm; P2Y₆ antagonist); MeSAMP (10 mM; P2Y₁₂ antagonist); TNP-ATPhydrate (25 mM; P2X₄ antagonist), and oxidized ATP (10 mM; P2X₇antagonist).

FIG. 4 depicts the process lengths of microglia extending to contact theinjured glial limitans following transcranial administration ofpurinergic receptor antagonists to mice subjected to meningealcompression injury.

FIG. 5 depicts the number of neutrophils following transcranialadministration of P2X₇ antagonist as a function of time in micesubjected to meningeal compression injury.

FIG. 6 depicts the ratio of the fluorescence of dye SR101 present in theparenchyma before and after pretreatment of the mice with vehicle (blackbar) or glutathione (white bar), wherein the mice were subjected tomeningeal compression injury.

FIG. 7 depicts the number of necrotic cells in the parenchyma of micesubjected to meningeal compression injury when the mice were treatedwith vehicle (black bar) and glutathione (gray bar, 15 min after injury,and white bar, 3 hrs after injury).

FIG. 8 depicts the number of neutrophils recruited to site of meningealcompression injury wherein the mice were pretreated by transcranialadministration of vehicle (black bar) and glutathione (white bar) andthen subjected to meningeal compression injury.

FIG. 9 depicts the percentage of microglia with jellyfish morphologypresent in the brain of mice subjected to meningeal compression injury,wherein the mice were pretreated with control (black bar) or glutathione(white bar).

FIG. 10 depicts the process of length of microglia contacting the gliallimitans in the brain of mice subjected to meningeal compression injury,wherein the mice were pretreated with vehicle (black bar) or glutathione(gray bar). The white bar represents the process length in uncompressedmice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relies, at least in part, on the ability of thecompounds of the invention to pass through the skull bone takingadvantage of the porous nature of the skull bone. All three layers ofthe skull bone, the upper cortical, the cancellous, and the lowercortical layers are porous to varying degrees. The cancellous bone isknown to be highly porous. As a result, compounds can be applied to theskull bone, and they can reach the CNS. In accordance with the presentinvention, the compounds can diffuse directly through the skull boneitself, especially if a compound is applied directly to or injected intothe skull bone. The skull of a mammal has anatomical features that allowdelivery of therapeutic agents to the CNS effectively and rapidly.

In an embodiment, when a compound of the invention is applied to thescalp, the compound may access the emissary veins. The emissary veinsdraining blood from extracranial sites into the intracranial sinusespierce a series of foramina present in the cranial bones. Scalp veinscommunicate with the sinuses of the brain via emissary veins. There arethirteen emissary veins connecting extracranial sites of the human headwith intracranial sinuses. Seven major sinuses within the skull areinter-connected by a number of anastomizing veins, which finally drainintracranially into jugular veins giving ample scope for the diffusionof the drug molecules into the nerve tissue of the brain.

Accordingly, in accordance with an embodiment, the invention provides amethod of treating or preventing a disease or disorder of the centralnervous system (CNS) in a patient comprising administeringtranscranially an effective amount of a reactive oxygen scavenger or ananti-inflammatory agent to the patient. The disease or disorder of theCNS can be any suitable disease or disorder, for example, those selectedfrom the group consisting of brain injury, inflammation, infection,degeneration of brain cells, stroke, brain edema, tumor, Alzheimer'sdisease, Parkinson's disease, and multiple sclerosis. In a particularembodiment, the disease or disorder of the CNS is brain injury, and moreparticularly traumatic brain injury (TBI).

In accordance with another embodiment, the invention provides a methodof inhibiting, reducing, or eliminating the formation of reactivemicroglia in a patient suffering from a traumatic brain injurycomprising administering transcranially an effective amount of ananti-inflammatory agent to the patient. In a further embodiment, theinvention provides a method of inhibiting, reducing, or eliminating therecruitment of neutrophils and/or monocytes in a patient suffering froma traumatic brain injury comprising administering transcranially aneffective amount of an anti-inflammatory agent to the patient. In yetanother embodiment, the invention provides a method of reducing thenumber of dead cells in the brain parenchyma or meninges in a patientsuffering from a traumatic brain injury comprising administeringtranscranially an effective amount of an anti-inflammatory agent to thepatient.

The CNS disease or disorder, e.g., TBI, can arise from activityassociated with boxing, football, soccer, hockey, armed conflict, orbrain surgery.

The various changes occurring in the CNS, particularly brain, which hasbeen injured or a CNS, particularly brain, undergoing treatment can beassessed by the imaging techniques described in Example 1.

Additionally, the presence of traumatic brain injury in a patient can beassessed by standard techniques used by a physician of skill in the art.These include, among others, Glasgow Coma Scale, which is a 15-pointtest that helps assess the severity of a brain injury by checkingpatient's ability to follow directions, to blink the eyes or to moveextremities; brain imaging techniques, including computer assistedtomography (CAT) scans, which allow visualization of fractures andevidence of bleeding in the brain (hemorrhage), large blood clots(hematomas), bruised brain tissue (contusions), and brain tissueswelling. In embodiments, the brain imaging technique used can bemagnetic resonance imaging (MRI), including Susceptibility WeightedImages (SWI), a sensitive method for detecting small hemorrhages in thebrain, and Diffusion tensor imaging (DTI), which consists of a minimumof six scans with diffusion gradients placed in an orthogonal manner. Insome embodiments, traumatic brain injury can be assessed by measuringintracranial pressure, which can occur by swelling of the brain.

Since neurobehavioral, particularly cognitive related, problems are amajor effect of traumatic brain injury, various methods used to assesscognitive function can be used. Such assessments include, among others,the following: Clinical Dementia Rating Scale (CDR), a dementia staginginstrument that classifies cognitive impairment along a continuum fromnormal aging to mild cognitive impairment to all stages of dementiaseverity; Folstein Mini-Mental State Exam (MMSE), which is commonly usedto measure of orientation and gross cognitive functioning used byphysicians and healthcare providers to screen for cognitive decline; andAlzheimer's Disease Assessment Scale-Cognitive (ADAS-C), a test commonlyused in detection of dementia and mild cognitive impairment.

Additional methods for assessing cognitive impairment from traumaticbrain injury can include, among others, various neuropsychological test,such as the following: Wechsler Test of Adult Reading (WTAR), which is ameasure of word pronunciation and is a reliable predictor of pre-morbidgeneral intellectual function; Wechsler Adult Intelligence Scale-3(WAIS-3)-Kaufman tetrad short form, which is used to measure generalintellectual functioning; Repeatable Battery for the Assessment ofNeuropsychological Status (RBANS), a comprehensive but relatively rapid,standardized measure of neurocognitive functioning in multiple domains,including memory, attention, language, and visuospatial/constructionalfunctions; Trailmaking Test Part A (Trails A), a widely-used measure ofcognitive processing and visuomotor speed, and with Part B, alsopreviously employed in studies of mild cognitive impairment (MCI);Trailmaking Test Part B (Trails B), a more complex measure of cognitiveprocessing with executive demands related to mental flexibility andworking memory; Controlled Oral Word Association Test (COWAT), awell-known measure of phonemically-controlled verbal fluency, sensitiveto cognitive slowing and impairments of executive functioning androutinely employed in dementia assessment and MCI studies; Boston NamingTest (BNT), a visual confrontation naming measure utilized to detectanomia or word-finding difficulties, which are common hallmarks ofcognitive decline in elderly populations with mild cognitive impairmentor early dementia; Automated Neuropsychological Assessment Metrics(ANAM), a computerized test designed to assess several cognitive domainsknown to be sensitive to change following concussion, includingattention and concentration, reaction time, working memory, new learningand memory, and speed of information processing; and SF-36, whichmeasures eight domains of health, including, physical functioning, rolelimitations due to physical health, bodily pain, general healthperceptions, vitality, social functioning, role limitations due toemotional problems, and mental health.

The various processes that undergo in the CNS after an injury can bequite complex. For example, in TBI, particularly in meningealcompression injury, the meningeal macrophages undergo rapid necrosis,releasing their contents into the subarachnoid space. This mediates abreach in the glial limitans, which in turn, triggers transformation ofthe underlying parenchymal microglia into massive phagocytic machineswith a jellyfish-like morphology. These microglia migrate to thecompromised glial limitans and appear to form a phagocytic barrierbetween the meninges and brain parenchyma. The phagocytic microglia arealways associated with areas of barrier compromise despite the continuedsurvival of astrocytes comprising glial limitans. The microglialresponse to meningeal compression is immediate and is followed shortlythereafter by swarms of neutrophils that invade the injury site.

The present invention further provides a kit or package comprising atleast one anti-inflammatory agent and printed materials containinginstructions for transcranially administering an anti-inflammatory agentto the patient having a disease or disorder of the central nervoussystem (CNS). The kit or package can further include a device foradministering the anti-inflammatory agent to the head, scalp, or skullbone of the patient. The kit or package can contain one dosage form, ormore than one dosage form, i.e., multiple dosage forms. If multipledosage forms are present in the kit or package, the multiple dosageforms can be optionally arranged for sequential administration. The kitor package can contain dosage forms of a sufficient number for 1, 2, 3,4, or more weeks, or months, of daily or weekly administration of theanti-inflammatory agent.

The kit or package can optionally include instructions with dosageforms. Such instructions can be in a form prescribed by a governmentagency regulating manufacture, use or sale of pharmaceutical products,which notice reflects approval by the agency of the manufacture, use orsale for human administration to treat the disease or disorder. Theinstructions can be in any form that conveys information on the use ofthe dosage forms in the kit or package according to the methodsdescribed herein. By way of example, the instructions can be in the formof a printed matter, or in the form of a pre-recorded media device.

In any of the above embodiments, the brain injury involves meningealcompression injury.

In any of the above embodiments, the ROS or anti-inflammatory agent isselected from the group consisting of antioxidants, purinergic receptorinhibitors, and connexin hemichannel inhibitors.

The ROS or anti-inflammatory agent can have any suitable molecularweight, preferably a low molecular weight compound. For example, themolecular weight, particularly the number average molecular weight, ofthe anti-inflammatory agent can be up to about 40,000 Daltons,particularly from about 300 to about 10,000 Daltons, and moreparticularly from about 300 to about 1000 Daltons. In an embodiment, theanti-inflammatory agent has a molecular weight, e.g., number averagemolecular weight, of about 600 to 1000 Daltons. In another embodiment,the anti-inflammatory agent has a molecular weight, e.g., number averagemolecular weight, of about 400 to 500 Daltons.

In embodiments, the ROS or anti-inflammatory agent passes through theskull by a diffusion mechanism. Thus, smaller molecular weight agentsreach the desired site of action more quickly than larger molecularweight agents.

In a particular embodiment, the ROS or anti-inflammatory agent is anantioxidant, for example, an antioxidant selected from the groupconsisting of glutathione, ascorbic acid, lipoic acid, uric acid,carotenes, α-tocopherol, ubiquinols, and combinations thereof, and moreparticularly, glutathione.

In an embodiment, the ROS or anti-inflammatory agent or antioxidant isnot docosahexaenoic acid or eicosapentaenoic acid or salts or estersthereof.

In accordance with an embodiment, the ROS or anti-inflammatory agent isa purinergic receptor inhibitor, for example, a P2X or a P2Y receptorinhibitor. Examples of such inhibitors include P2X₄ receptor inhibitors,P2X₇ receptor inhibitors, P2Y₆ receptor inhibitors, and P2Y₁₂ receptorinhibitors.

In a particular embodiment, the inhibitor is a P2X₄ receptor inhibitor.Any suitable P2X₄ receptor inhibitor can be used. For example,trinitrophenyl-ATP hydrate can be used as a P2X₄ receptor inhibitor.

Examples of other P2X₄ receptor inhibitors include 1,4-diazepin-2-onederivatives disclosed in WO 2004/085440 A1 and US 2010/0256123 A1,piperazine derivatives disclosed in WO 2005/037803 A1, WO 2004/089915A1, and US 2011/0092703 A1, and tricyclic compounds disclosed in WO2007/072974 A1, WO 2007/074940 A1, and WO 2008/023847 A1. For example,the P2X₄ receptor inhibitor can be, as disclosed in WO 2004/085440 A1,of formula (A) shown below:

in which R₁ is halogen and R₂ is hydrogen, halogen, nitro, cyano,C(O)—OR₃, C(O)—NR₄R₅, SO₂—OR₃, or SO₂—NR₄R₆, or R₁ is hydrogen and R₂ ishalogen, nitro, cyano, C(O)—OR₃, C(O)—NR₄R₅, SO₂—OR₃ or SO₂—NR₄R₆.wherein R₃, R₄, and R₅ are hydrogen or alkyl.

In a particular embodiment, the inhibitor is a P2X₇ receptor inhibitor.Any suitable P2X₇ receptor inhibitor can be used. For example, oxidizedATP (o-ATP) can be used as a P2X₇ receptor inhibitor, which is a ribosering-opened dialdehyde analog of ATP. The oxidation of ATP can becarried out by a periodate.

Examples of other P2X₇ receptor inhibitors include the pyrazolederivatives disclosed in US 2007/0259920 A1, the amino-tetrazolederivatives disclosed in WO 2005/111,003 A1, as well as1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine,hexamethylene amiloride, and brilliant blue G. For example, a P2X₇receptor inhibitor, as disclosed in US 2007/0259920 A1, can be acompound of formula (I):

or a pharmaceutically acceptable salt, prodrug, salt of prodrug, or acombination thereof, whereinR₁ is hydrogen or —CN, and R₂ is hydrogen, orR₁ and R₂ together with the carbon atoms to which they are attached,form a monocyclic saturated ring consisting of 5, 6 or 7 carbon atomsand one of the carbon atoms of the ring is optionally replaced by aheteroatom selected from the group consisting of S, N, NH, O, SO andSO₂; and said ring is optionally substituted with 1 or 2 substituentsselected from the group consisting of alkyl, halogen, haloalkyl,—C(O)alkyl, and —S(O)₂alkyl;R₃ is halogen, —CN, haloalkyl, alkoxy or haloalkoxy;R₄ is alkyl, halogen, —CN, haloalkyl, alkoxy or haloalkoxy;R₅ is hydrogen, alkyl, halogen, —CN, haloalkyl, alkoxy or haloalkoxy;R₆ is —N(H)—W, or —N(H)—C(R_(x))(H)—W₁; whereinR_(x) is hydrogen, alkyl or haloalkyl,

W is

whereinA is a five or six membered monocyclic ring selected from the groupconsisting of cycloalkyl and heterocycle and is optionally substitutedwith 1, 2, or 3 substituents selected from the group consisting ofalkyl, halo and haloalkyl;B is phenyl or monocyclic heteroaryl, optionally substituted with 1, 2or 3 substituents selected from the group consisting of halo, alkyl,—CN, —OR_(A), —SR_(A), —N(R_(A))(R_(B)) and haloalkyl;q is 0 or 1;

R_(y) is X or -L-X;

W₁ is phenyl or monocyclic heteroaryl, wherein each W₁ is optionallyfused with a monocyclic, five or six-membered ring selected from thegroup consisting of phenyl, heteroaryl, heterocycle, cycloalkyl andcycloalkenyl; wherein each ring as represented by W₁ is independentlyunsubstituted, substituted with one, two or three R₇, or substitutedwith zero, one or two R₇ and one substituent selected from the groupconsisting of X and -L-X;L at each occurrence is independently O, N(H), N(alkyl), S, S(O), S(O)₂,S(O)₂N(H), SO₂N(alkyl), N(H)S(O)₂, N(alkyl)S(O)₂, CON(H), CON(alkyl),N(H)CO, or N(alkyl)CO);X, at each occurrence is independently aryl, heteroaryl, cycloalkyl,cycloalkenyl, or heterocycle; each of which is independentlyunsubstituted or substituted with one, two or three R₇;R₇ at each occurrence is independently alkyl, alkenyl, CN, NO₂, halo,══O, —OR_(A), —SR_(A), —S(O)R_(A), —S(O)₂R_(A), —S(O)₂N(R_(A))(R_(B)),—N(R_(A))(R_(B)), —C(O)R_(A), —C(O)OR_(A), —C(O)N(R_(A))(R_(B)),haloalkyl, -alkyl-OR_(A)-alkyl-SR_(A), -alkyl-S(O)R_(A),-alkyl-S(O)₂R_(A), -alkyl-S(O)₂N(R_(A))(R_(B)), -alkyl-N(R_(A))(R_(B)),-alkyl-C(O)R_(A), -alkyl-C(O)OR_(A), or -alkyl-C(O)N(R_(A))(R_(B)); andR_(A) and R_(B) at each occurrence are independently hydrogen, alkyl,alkenyl or haloalkyl.

In a particular embodiment, the inhibitor is a P2Y₆ receptor inhibitor.Any suitable P2Y₆ receptor inhibitor can be used. For example, N,N″-1,4-butanediylbis[N′-3-isothiocyanatophenyl)]thiourea (or MRS 2578)can be used as a P2Y₆ receptor inhibitor.

Examples of other P2Y₆ receptor inhibitors include the diisothiocyanatederivative of 1,2-diphenylethane (MRS 2567) and1,4-phenylendiisothiocyanate derivative MRS 2575, disclosed in Mamedova,L. K., et al., Biochemical Pharmacology, 67, 1763-1770 (2004).

In a particular embodiment, the inhibitor is a P2Y₁₂ receptor inhibitor.Any suitable P2Y₁₂ receptor inhibitor can be used. For example, MeSAMPcan be used as a receptor inhibitor.

Examples of other P2Y₁₂ receptor inhibitor include anthraquinonederivatives of formula (I) disclosed in US 2010/0210654 A1.

wherein:A and B are independently CH₂, O, S, NH, C═O, C═NH, C═S, or C═N—OH;X is selected from the group consisting of NH, O, S, C═O, and CH₂;R¹ and R² are independently selected from the group consisting ofhydrogen, unsubstituted or substituted C₁-C₁₀ alkyl, unsubstituted orsubstituted C₁-C₁₀ alkenyl, unsubstituted or substituted C₁-C₁₀ alkynyl,unsubstituted or substituted C₃-C₈ cycloalkyl, unsubstituted orsubstituted C₁-C₁₀ alkoxy, unsubstituted or substituted C₃-C₈cycloalkoxy, unsubstituted or substituted C₆-C₁₄ aryl, an unsubstitutedor substituted 5- to 10-membered heteroaryl wherein 1 to 4 ring atomsare independently selected from nitrogen, oxygen or sulfur, anunsubstituted or substituted 5- to 10-membered heteroalicyclic ringwherein 1 to 3 ring atoms are independently nitrogen, oxygen or sulfur,—OR, —C(O)R, —C(O)OR, —C(O)NRR′, —NRR′, —S(O)₂R, —S(O)₂OR, and—S(O)₂NRR′;R³ is selected from the group consisting of hydrogen, unsubstituted orsubstituted C₁-C₁₀ alkyl, unsubstituted or substituted C₁-C₁₀ alkenyl,unsubstituted or substituted C₁-C₁₀ alkynyl, unsubstituted orsubstituted C₃-C₈ cycloalkyl, unsubstituted or substituted C₁-C₁₀alkoxy, unsubstituted or substituted C₃-C₈ cycloalkoxy, unsubstituted orsubstituted C₆-C₁₄ aryl, an unsubstituted or substituted 5- to10-membered heteroaryl wherein 1 to 4 ring atoms are independentlyselected from nitrogen, oxygen or sulfur, an unsubstituted orsubstituted 5- to 10-membered heteroalicyclic ring wherein 1 to 3 ringatoms are independently nitrogen, oxygen or sulfur, alkylaryl,alkylheteroaryl, an unsubstituted or substituted 7 to 12-memberedbicyclic alkyl or heterocyclic ring wherein 1 to 3 ring members areindependently nitrogen, oxygen or sulfur, an unsubstituted orsubstituted 10 to 16-membered tricyclic alkyl or heterocyclic ringwherein 1 to 3 ring members are independently nitrogen, oxygen orsulfur,

R⁴ and R⁵ are hydrogen, or, combined, R⁴ and R⁵ may, together with thecarbon atoms to which they are attached, form a group selected of thegroups consisting of unsubstituted or substituted C₃-C₈ cycloalkyl,unsubstituted or substituted C₆-C₁₄ aryl, unsubstituted or substituted5- to 10-membered heteroaryl wherein 1 to 4 ring atoms are independentlyselected from nitrogen, oxygen or sulfur, and unsubstituted orsubstituted 5- to 10-membered heteroalicyclic ring wherein 1 to 3 ringatoms are independently nitrogen, oxygen or sulfur;R⁶ represents one to four substituents which are independently selectedfrom the group consisting of hydrogen, halogen, unsubstituted orsubstituted C₁-C₄ alkyl, alkenyl or alkynyl, an unsubstituted orsubstituted 5- to 10-membered heteroaryl wherein 1 to 4 ring atoms areindependently selected from nitrogen, oxygen or sulfur, an unsubstitutedor substituted 5- to 10-membered heteroalicyclic ring wherein 1 to 3ring atoms are independently nitrogen, oxygen or sulfur, —OR, —NRR′,—NO₂, unsubstituted or substituted C₁-C₄ alkoxy, —C(O)R, —C(O)OR,—C(O)NRR′, —S(O)₂R, —S(O)₂OR, and —S(O)₂NRR′;Y is selected from the group consisting of hydrogen, halogen, NH, O, S,CH₂, CH₂CH₂, C(O), C(O)O, S(O)₂O, or unsubstituted C₁-C₄ alkoxy, withthe proviso that when Y is hydrogen, halogen or alkoxy R⁷ is missing;R⁷ is selected from the group consisting of hydrogen, halogen, —OR,unsubstituted or substituted C₁-C₁₀ alkyl, alkenyl or alkynyl,unsubstituted or substituted C₁-C₁₀ alkoxy, unsubstituted or substitutedC₃-C₈ cycloalkoxy, unsubstituted or substituted C₃-C₈ cycloalkyl,unsubstituted or substituted C₆-C₁₄ aryl, an unsubstituted orsubstituted 5- to 10-membered heteroaryl wherein 1 to 4 ring atoms areindependently selected from nitrogen, oxygen or sulfur, an unsubstitutedor substituted 5- to 10-membered heteroalicyclic wherein 1 to 3 ringatoms are independently nitrogen, oxygen or sulfur, —C(O)R, —C(O)OR,—C(O)NRR′, —NRR′, —S(O)₂R, —S(O)₂OR, —S(O)₂NRR′, and —P(O)RR′; andR and R′ are independently selected from the group consisting ofhydrogen and unsubstituted C₁-C₄ alkyl.

In a particular embodiment, the ROS or anti-inflammatory agent is aninhibitor of a connexin hemichannel. Any suitable connexin hemichannelreceptor inhibitor can be used. For example,(3β)-3-[(3-carboxypropanoyl)oxy]-11-oxoolean-12-en-30-oic acid (orCarbenoxolone) can be used as an inhibitor of a connexin hemichannel.

Examples of other connexin hemichannel inhibitors include the antisenseoligodeoxynucleotides disclosed in U.S. Pat. No. 7,098,190, for example,see col. 2, lines 1-32.

Topical administration includes direct application of the drug to theskull of the patient through the scalp. Such administration couldinvolve rubbing the formulation onto the top of the head, providing atransdermal patch on the head, and subcutaneous injection under thescalp. For example, the hair of the patient's scalp can be removed ortrimmed without injuring the skin. A portion of the topical formulationis applied to the scalp by a dropper or other applicator and theformulation allowed to penetrate the skin. A second portion and a thirdportion of the topical formulation can then be applied to the scalp insuccession. Optionally, between each application, the scalp can berubbed to enhance the absorption of the formulation. The rubbing can beperformed manually or by an automated device such as a stroking device.

In accordance with the invention, the compounds of the invention couldbe directly applied to the exposed skull bone, for example, as couldoccur following injury or surgery. In addition, or alternatively, thecompounds could be injected directly into the skull bone itself. Inmammals, there are 3 layers of skull bone. The first layer is referredto as the upper cortical bone. This bone is denser than the middle layeror the cancellous bone. The cancellous bone is quite porous.Accordingly, advantageously, compounds of the invention can beadministered, e.g., injected, directly into the cancellous bone. Thecompounds would then diffuse more quickly through the lower corticallayer of bone directly into the underlying meninges. In rodents,compounds in accordance with an embodiment of the invention applieddirectly to the upper cortical bone pass through all three layers.Compounds having a suitable molecular weight have the ability to passdirectly through the skull. The compounds can be applied topically tothe scalp; however, following injury or during surgery, compounds couldbe applied directly to the skull bone itself. In an embodiment, theskull bone could be removed, for example, to relieve the pressure due tosevere TBI, and the compound can be administered to the injured brain.

Topically applied compositions are generally in the form of liquids,creams, pastes, lotions and gels, film, foil, paint, suspension,ointment, solution, drop, swab, infusion, or sprays. In someembodiments, the composition contains at least one active component anda suitable vehicle or carrier. It may also contain other components,such as an anti-irritant. The carrier can be a liquid, solid orsemi-solid. In embodiments, the composition is an aqueous solution.Alternatively, the composition can be a dispersion, emulsion, gel,lotion or cream vehicle for the various components. In one embodiment,the primary vehicle is water or a biocompatible solvent that issubstantially neutral or that has been rendered substantially neutral.The liquid vehicle can include other materials, such as buffers,alcohols, glycerin, and mineral oils with various emulsifiers ordispersing agents as known in the art to obtain the desired pH,consistency and viscosity. It is possible that the compositions can beproduced as solids, such as powders or granules. The solids can beapplied directly or dissolved in water or a biocompatible solvent priorto use to form a solution that is substantially neutral or that has beenrendered substantially neutral and that can then be applied to thetarget site. In embodiments of the invention, the vehicle for topicalapplication to the skin can include water, buffered solutions, variousalcohols, glycols such as glycerin, lipid materials such as fatty acids,mineral oils, phosphoglycerides, collagen, gelatin and silicone basedmaterials. In an embodiment, the therapeutic agent can be applied bysubcutaneous injection under the scalp or directly into the skull boneitself.

In an embodiment, the topical formulation can be applied as a sustainedrelease gel or transdermal patch.

Optionally, in addition to the topical administration described above,the antioxidant can also be administered to the patient via other modesof administration. Thus, multi-modal administration is also contemplatedwithin the scope of the invention so long as a topical administration isinvolved.

The antioxidant can be administered in combination with apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier (or excipient) is preferably one that is chemically inert to thecompound of the invention and one that has no detrimental side effectsor toxicity under the conditions of use. Such pharmaceuticallyacceptable carriers preferably include saline (e.g., 0.9% saline),Cremophor EL (which is a derivative of castor oil and ethylene oxideavailable from Sigma Chemical Co., St. Louis, Mo.) (e.g., 5% CremophorEL/5% ethanol/90% saline, 10% Cremophor EL/90% saline, or 50% CremophorEL/50% ethanol), propylene glycol (e.g., 40% propylene glycol/10%ethanol/50% water), polyethylene glycol (e.g., 40% PEG 400/60% saline),and alcohol (e.g., 40% ethanol/60% water). A preferred pharmaceuticalcarrier is polyethylene glycol, such as PEG 400, and particularly acomposition comprising 40% PEG 400 and 60% water or saline. The choiceof carrier will be determined in part by the particular compound chosen,as well as by the particular method used to administer the composition.Accordingly, there is a wide variety of suitable formulations of thepharmaceutical composition of the present invention.

The following formulations for oral, aerosol, parenteral, subcutaneous,intravenous, intraarterial, intramuscular, interperitoneal, rectal, andvaginal administration are merely exemplary and are in no way limiting.The pharmaceutical compositions can be administered parenterally, e.g.,intravenously, intraarterially, subcutaneously, intradermally,intrathecally, or intramuscularly. Thus, the invention providescompositions for parenteral administration that comprise a solution ofthe compound of the invention dissolved or suspended in an acceptablecarrier suitable for parenteral administration, including aqueous andnon-aqueous, isotonic sterile injection solutions.

Overall, the requirements for effective pharmaceutical carriers forparenteral compositions are well known to those of ordinary skill in theart. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986). Such compositions include solutions containing anti-oxidants,buffers, bacteriostats, and solutes that render the formulation isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. The compound can beadministered in a physiologically acceptable diluent in a pharmaceuticalcarrier, such as a sterile liquid or mixture of liquids, includingwater, saline, aqueous dextrose and related sugar solutions, an alcohol,such as ethanol, isopropanol (for example in topical applications), orhexadecyl alcohol, glycols, such as propylene glycol or polyethyleneglycol, dimethylsulfoxide, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils useful in parenteral formulations include petroleum, animal,vegetable, and synthetic oils. Specific examples of oils useful in suchformulations include peanut, soybean, sesame, cottonseed, corn, olive,petrolatum, and mineral oil. Suitable fatty acids for use in parenteralformulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acidesters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylene polypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations typically will contain from about 0.5% orless to about 25% or more by weight of a compound of the invention insolution. Preservatives and buffers can be used. In order to minimize oreliminate irritation at the site of injection, such compositions cancontain one or more nonionic surfactants having a hydrophile-lipophilebalance (HLB) of from about 12 to about 17. The quantity of surfactantin such formulations will typically range from about 5% to about 15% byweight. Suitable surfactants include polyethylene sorbitan fatty acidesters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of a compound of the inventiondissolved in diluents, such as water, saline, or orange juice; (b)capsules, sachets, tablets, lozenges, and troches, each containing apre-determined amount of the compound of the invention, as solids orgranules; (c) powders; (d) suspensions in an appropriate liquid; and (e)suitable emulsions. Liquid formulations can include diluents, such aswater and alcohols, for example, ethanol, benzyl alcohol, and thepolyethylene alcohols, either with or without the addition of apharmaceutically acceptable surfactant, suspending agent, or emulsifyingagent. Capsule forms can be of the ordinary hard- or soft-shelledgelatin type containing, for example, surfactants, lubricants, and inertfillers, such as lactose, sucrose, calcium phosphate, and cornstarch.Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible excipients. Lozenge forms cancomprise the compound ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising a compound of theinvention in an inert base, such as gelatin and glycerin, or sucrose andacacia, emulsions, gels, and the like containing, in addition to thecompound of the invention, such excipients as are known in the art.

Additionally, the antioxidants can be made into suppositories by mixingwith a variety of bases, such as emulsifying bases or water-solublebases. Formulations suitable for vaginal administration can be presentedas pessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the compound ingredient, such carriers as areknown in the art to be appropriate.

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop. As used herein, the term “ameliorating,” with reference to adisease or pathological condition, refers to any observable beneficialeffect of the treatment. The beneficial effect can be evidenced, forexample, by a delayed onset of clinical symptoms of the disease in asusceptible subject, a reduction in severity of some or all clinicalsymptoms of the disease, a slower progression of the disease, animprovement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. The phrase “treating a disease” refers to inhibitingthe full development of a disease or condition, for example, in asubject who is at risk for a disease such as cancer, particularly ametastatic cancer.

A typical pharmaceutical composition for intravenous infusion could bemade up to contain 250 ml of sterile Ringer's solution, and 100 mg of atleast one compound of the invention. Actual methods for preparingparenterally administrable compounds of the invention will be known orapparent to those skilled in the art and are described in more detailin, for example, Remington's Pharmaceutical Science (17^(th) ed., MackPublishing Company, Easton, Pa., 1985).

It will be appreciated by one of ordinary skill in the art that, inaddition to the aforedescribed pharmaceutical compositions, theanti-inflammatory agent or compound of the invention can be formulatedas inclusion complexes, such as cyclodextrin inclusion complexes, orliposomes. Liposomes can serve to target a compound of the invention toa particular tissue, such as lymphoid tissue or cancerous hepatic cells.Liposomes can also be used to increase the half-life of a compound ofthe invention. Many methods are available for preparing liposomes, asdescribed in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9,467 (1980) and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369.

The treatment regimens can vary depending on the severity of the CNSdisease or disorder. For example, particularly for treating TBI, in anembodiment, the subject is treated within at least 1 hr, within at least2 hr, or within at least 6 hr of suffering the CNS disease or disorder.In another embodiment, the subject is treated for at least 7 days, atleast 14 days, or at least 28 days after suffering the CNS disease ordisorder.

The therapeutically effective amount of the compound or compoundsadministered can vary depending upon the desired effects and the factorsnoted above. IN accordance with an embodiment, examples of drug dosagescan be between 0.01 mg/kg and 250 mg/kg of the subject's body weight,and more typically between about 0.05 mg/kg and 100 mg/kg, such as fromabout 0.2 to about 80 mg/kg, from about 5 to about 40 mg/kg or fromabout 10 to about 30 mg/kg of the subject's body weight, per day. Inembodiments, the drug dosages can be between 3 mg/kg and 85 mg/kg of thesubject's body weight, or between about 3 mg/kg and 60 mg/kg, such asfrom about 5 mg/kg to about 60 mg/kg, from about 10 to about 60 mg/kg orfrom about 20 to about 60 mg/kg of the subject's body weight, per day.

Unit dosage forms can be formulated based upon the suitable rangesrecited above and the subject's body weight. The term “unit dosage form”as used herein refers to a physically discrete unit of therapeutic agentappropriate for the subject to be treated.

Alternatively, dosages are calculated based on body surface area andfrom about 1 mg/m² to about 200 mg/m², such as from about 5 mg/m² toabout 100 mg/m² will be administered to the subject per day. Inparticular embodiments, administration of the therapeutically effectiveamount of the compound or compounds involves administering to thesubject from about 5 mg/m² to about 50 mg/m², such as from about 10mg/m² to about 40 mg/m² per day. It is currently believed that a singledosage of the compound or compounds is suitable, however atherapeutically effective dosage can be supplied over an extended periodof time or in multiple doses per day. Thus, unit dosage forms also canbe calculated using a subject's body surface area based on the suitableranges recited above and the desired dosing schedule.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1 Materials and Methods

Mice.

C57BL/6J (B6) and B6.129P-Cx3cr1^(tm1Ltt)/J (CX3CR1^(gfp/gfp)) mice wereobtained from The Jackson Laboratories. CX3CR1^(gfp/+) mice weregenerated by crossing B6 mice with CX3CR1^(gfp/gfp) mice in a closebreeding facility at the National Institutes of Health (NIH). B6LysM-GFP heterozygous knock-in mice (LysM^(gfp/+)) were generouslyprovided by Dr. Thomas Graf (Albert Einstein College of Medicine) (PMID:10887140) and maintained at the NIH. All mice were housed under specificpathogen-free conditions and treated in accordance with InstitutionalAnimal Care and Use Committee at the NIH.

Skull Thinning & Compression Injury.

For imaging experiments, mice were anesthetized with ketamine (85mg/kg), xylazine (13 mg/kg), and acepromazine (2 mg/kg) in PBS andmaintained at a core temperature of 37° C. The skull bone over thebarrel cortex was then thinned to a thickness of ˜30-40 μm as described(PMID: 20134419). For this procedure, the bone was manually thinned overa 30 minute period, and the amount of downward pressure was kept to aminimum. To induce a compression injury, the skull was thinned within 5min to a thickness of ˜10-15 μm. Once thinned, the blunt of a surgicalinstrument was used to gently press the pliable skull bone downward.This resulted in the skull bone collapsing (without breaking) inwardtoward the surface of the brain. In one set of experiments, the skullbone was intentionally cracked to induce a severe injury. Imaging wasperformed immediately after injury.

Intravital Two-Photon Laser Scanning Microscopy.

Mice with a normally thinned or compressed skull bone were imaged usinga Leica SP5 two-photon imaging system (Leica Microsystems, Bannockburn,Ill.) equipped with an 8000 Hz resonant scanner, a 20×/1.0 NA dippingobjective, and two Mai Tai HP DeepSee Lasers (SpectraPhysics) tuned to905, 920 or 970 nm. Fluorescence emission was separated by highefficiency custom dichroic mirrors (Semrock) and collected with a NDD4external detector (Leica). Stacks of images were acquired using a stepsize of 1.0 μm to a depth of 150 μm. Time lapse movies were acquiredwith 1 min intervals between 3D stacks. For all imaging studies, the 20×lens was submerged directly into artificial cerebral spinal fluid (aCSF;119 mM NaCl, 26.2 mM NaHCO₃, 2.5 mm KCl, 1 mM NaH₂PO₄, 1.3 mM MgCl₂, 1.2mM CaCl₂, 0.4% glucose, pH 7.4) placed atop the thinned skull.

Fluorescent Dyes.

To visualize brain vasculature, mice were injected intravenously (i.v.)10 min prior to imaging with 50 μl Qtracker 655 nm non-targeted quantumdots in PBS (0.2 uM; Invitrogen). Cell death was visualized byincubating the thinned skull with propidium iodide (1.5 mM) in aCSF for30 min. This was followed by a single wash with aCSF and then imaging.Reactive oxygen species (ROS) were visualized by applying Amplex Red(500 uM) transcranially for 10 min. This was followed by immediateimaging. Astrocytes were visualized by transcranially applying SR101 (10mM) for 30 min. This was followed by a 1 hr aCSF wash and then imaging.

Purinergic Receptor, Connexin Hemichannel, and ROS Antagonism.

Prior to skull thinning, antagonists diluted in aCSF were applieddirectly to the skull bone, and vehicle was simultaneously applied tothe opposite hemisphere to serve as a control. The following antagonistsfrom Sigma were used: TNP-ATP hydrate (antagonist of the P2X₄ receptor;25 mM), oxidated ATP (P2X₇; 10 mM), MRS2578 (P2Y6; diluted a 50 mM DMSOstock 1:100 in aCSF to a final concentration of 500 uM), MeSAMP (P2Y12;10 mM), carbenoxelone (CBX) (connexin hemichannels; 100 mM), andglutathione (ROS; 100 mM). Vehicle and purinergic receptor antagonistswere applied as a 3 mm diameter bubble on the skull surface andreplenished as needed over a 30 min incubation period to prevent drying.This allowed the antagonists to continuously pass through the skullbone. Following the 30 min incubation, the skull was dried and thenthinned to induce a compression injury over both hemispheres. Imagingwas initiated immediately after injury. For most studies, the skull bonepre-incubated with antagonists was imaged continuously for 3-10 hrs,after which a 3D stack was captured from both the vehicle and antagonisttreated areas for quantitative purposes. Glutathione was also applied at15 min and 3 hrs following compression injury to determine the impact oncell death. For these studies, glutathione was added directly to theaCSF submerging solution (100 mM) while imaging. The glutathione wasmaintained in the submerging solution for the entire imaging experiment.A similar study was conducted using 10 mM CBX.

Glial Limitans Leakage Assay.

For permeability studies, areas of skull bone were pre-incubated withvehicle, CBX (100 mM), or glutathione (100 mM) for 30 min as describedabove. Afterward, a meningeal compression injury was induced. Whileimaging, antagonists (10 mM CBX or 100 mM glutathione) were addeddirectly to the aCSF submerging solution. After 3 hrs of imaging, SR101(1 mM) was applied for 15 minutes, followed by a 30 min aCSF wash. A 3Dstack was then captured to quantify the degree of SR₁₀₁ leakage throughthe glial limitans.

Skull Bone Permeability Analysis.

For skull bone permeability studies, the following rhodamine-labeleddextrans were placed directly on the intact skull bone for up to 30 min:3,000 MW (25 mM), 10,000 MW (5 mM), 40,000 MW (1 mM), and 70,000 MW (0.5mM). SR101 (1 mM), a sulforhodamine dye, was used as a representative of600 MW compound. Compounds were replenished as needed to prevent drying.Following the incubation period, the skull bone was quickly thinned andimaged. A 3D stack was captured to determine if the fluorescence couldbe found beneath the skull bone.

Image Analysis.

All quantitative analyses and processing of 3D/4D imaging data wereperformed using Imaris 7.0 software (Bitplane). Supplemental movies wereconstructed and annotated using Adobe Premiere Pro CS4. Microglia“honeycomb” reactions were quantified from 434×434×150 μm (xyz) 3D imagestacks obtained at selected time points in CX3CR1^(gfp/+) mice. This wasaccomplished by measuring the total length of microglial processes incontact with the glial limitans. Microglial cell bodies were firstselected using the Imaris “spots” tool. Afterward, 10 microglia permouse were randomly selected for quantification of process length. Onlycells with all of their processes in the field of view and no more than50 μm beneath the skull bone were quantified. Microglial processes thatwere touching (flat against) the glial limitans were labeled andmeasured using the Imaris “filaments” tool. Data were then representedon a per cell basis as the length of microglial processes in contactwith the glial limitans. Microglia with a “jellyfish” morphology wereidentified as those having processes greater than 20 μm in diameter. Thenumber of “jellyfish” microglia was then divided by the total number ofmicroglia within 50 μm of the skull bone and multiplied by 100 togenerate a percentage. To quantify cell death, propidium iodide-positivecells were labeled in 3D stacks using the Imaris “spots” tool. Deadcells from 0 to 5 μm below the compressed skull were consideredmeningeal, whereas cells from 5 to 100 μm were considered parenchymal.The number of dead cells was divided by volume analyzed and representedas cells per mm³. Neutrophils were quantified in LysM^(GFP)/+ mice usingthe Imaris “spots” tool. Following compression injury, neutrophils werenever observed in the brain parenchyma, and, therefore, were quantifiedonly in the meningeal space (0 to 5 μm below the compressed skull). Thenumber of neutrophils was divided by the volume analyzed and representedas cells per mm³. To quantify leakage of SR101 through the gliallimitans, a 50×50×100 μm (xyz) solid box was generated using the Imaris“surfaces” tool. The box was placed 25 μm beneath the epicenter of thecompression injury (i.e. the lowest point of the compressed skull bone).The mean fluorescent intensity of SR101 signal inside of this box wasthen calculated. The value obtained beneath the antagonist treated skull(glutathione or CBX) was divided by the vehicle control area (from theopposite hemisphere) to generate a fluorescence ratio.

Example 2

This example illustrates effects of meningeal compression injury in thebrain. Mice were anesthetized with ketamine, xylazine, and acepromazine.While under anesthesia, skulls were thinned surgically to a thickness ofa ˜15 microns and then lightly pressed downward using a blunt objectuntil the skull bone collapsed, thereby producing a meningealcompression injury. At selected time points following compressioninjury, 1.5 mM propidium iodide in artificial cerebral spinal fluid(aCSF) was applied for 30 minutes transcranially. This was followed by asingle wash with aCSF and then imaged intravitally. For all imagingexperiments, a Leica SP5 two photon (2P) microscope fitted with two MaiTai DeepSee lasers, a 20× (1.0 N/A) objective, a resonant scanner, and aquad external detector array, was utilized. The standard dimensions of a3D data set were 434 μm×434 μm×100 μm (xyz) with a z step size of 1 μm.For 4D movies, z stacks were collected every 30 seconds. All data setswere analyzed using 4D image analysis software (Imaris). Dead cells wereidentified as being propidium iodide positive. Cells located from 0 to 5μm below the skull bone were considered meningeal, whereas cells from 5to 100 μm were considered parenchymal. As shown in FIG. 1, meningealdeath occurred immediately after meningeal compression injury.Parenchymal death occurred 9 hours post compression injury.

Example 3

This example illustrates the effect of molecular weight of the compoundson the rate of transcranial permeation. To define the time and size ofcompounds that pass directly through the intact (non-thinned) skull, 10mM SR101 (600 MW) or different sized rhodamine-dextrans (25 mM 3,000 MW;5 mM 10,000 MW; 1 mM 40,000 MW; 0.5 mM 70,000 MW) were applied to theskull bone. A 3 mm diameter bubble was left on the skull surface for theamount of time noted in minutes on the y axis of the table above.Because all compounds fluoresced, 2P microscopy was performedimmediately after transcranial loading to determine if the compoundswere present in the subarachnoid space. The results obtained are shownin FIG. 2, where white indicates that a compound was found in thesubarachnoid space, whereas black indicates that it was not. Grayindicates that the compound was not tested at the indicated time. Smallcompounds (e.g. SR101) pass through the skull more quickly than largercompounds (e.g. 40K MW dextran). 70K MW dextran was not able to passthrough the skull in 30 min.

Example 4

This example illustrates the effect of certain embodiment purinergicreceptor antagonists in reducing the formation of phagocytic microgliawhen transcranially administered to the skull. The following purinergicreceptor antagonists were applied directly to the skull bone in aCSF 30min prior to inducing a meningeal compression injury: MRS2578 (500 μM;P2Y₆ inhibitor), MeSAMP (10 mM; P2Y₁₂ inhibitor), TNP-ATP hydrate (25mM; P2X₄ inhibitor), and oxidized ATP (10 mM; P2X₇ inhibitor). aCSF wasused as a vehicle control and placed on the skull bone of the oppositehemisphere. After the 30 min incubation, a meningeal compression injurywas induced beneath the vehicle and drug-treated portions of skull bone.This experiment was performed in heterozygous CX3CR1-GFP mice tofacilitate imaging of microglia by 2P microscopy. Phagocytic microgliawith processes greater than 20 μm in diameter (referred to as thejellyfish microglia) were quantified 3 hrs post-injury. As shown in FIG.3, transcranial administration of P2Y₆, P2Y₁₂, and P2X₄ (but not P2X₇)antagonists all significantly reduced (asterisks, p<0.05) the percentageof phagocytic microglia in the imaging field.

Three hours following injury, the length of microglia processesassociated with the glial limitans were quantified from 2P microscopyimages. Transcranial administration of P2X₄ and P2Y₁₂ (but not P2X₇ andP2Y₆) antagonists significantly reduced (asterisks, p<0.05) the lengthof microglial processes that were in contact with the glial limitans, asshown in FIG. 4.

FIG. 5 illustrates that the transcranial administration of a P2X₇antagonist prevents neutrophil recruitment following meningealcompression injury. A P2X₇ antagonist (oxidated ATP) was applied to theskull of a LysM-GFP transgenic reporter mouse as described above (seeExample 4). Monocytes and neutrophils are fluorescently tagged inLysM-GFP mice and can be distinguished from one another based on theirfluorescent intensity. Following meningeal compression, neutrophils arerecruited to the injury site within 3 hrs. This recruitment iscompletely blocked (asterisks, p<0.05) by transcranial administration ofa P2X₇ antagonist.

Example 5

This example illustrates the effect of glutathione in treating meningealcompression injury. As shown in FIG. 6, transcranial administrationglutathione reduces the number of dead cells in the brain parenchyma. A100 mM solution of glutathione (a reactive oxygen species scavenger) inaCSF was applied transcranially at 15 min or 3 hrs following meningealcompression and maintain until 12 hrs post-injury, at which pointpropidium iodide was added to label dead cells. The number of dead cellsin the brain parenchyma was quantified as described in Example 2.Initiation of glutathione treatment at 15 min and 3 hrs post-injurysignificantly reduced (asterisks, p<0.05) the number of dead cells inthe brain parenchyma.

Example 6

This example illustrates that transcranial administration of glutathionereduces breakdown of glial limitans. Meningeal compression inducedbreakdown of the glial limitans, which is the border between themeninges and brain parenchyma. The glial limitans breakdown wasquantified by administering a fluorescent dye (SR101) transcranially for15 min (see FIG. 2). When the glial limitans membrane was intact, SR101remains entirely in the meningeal space; however, SR101 leaked into thebrain parenchyma when the glial limitans was damaged. Pretreatment with100 mM glutathione (Example 4) significantly reduced (asterisks, p<0.05)the leakage of SR101 into the brain parenchyma when compared to thevehicle control group.

Further, as shown in FIG. 8, transcranial administration of glutathionelimits neutrophil recruitment. As noted above (see FIG. 5), neutrophilsare recruited to the site of a meningeal compression injury.Pretreatment with 100 mM glutathione (Example 4) significantly reduced(asterisks, p<0.05) the recruitment of neutrophils following compressioninjury. The reduced inflammation stems from the decrease in cellularinjury (see FIG. 6).

Furthermore, as shown in FIG. 9, transcranial administration ofglutathione prevents formation of phagocytic microglia. As noted above,phagocytic microglia are generated following a meningeal compressioninjury. These cells participate in the cleanup of cellular debris.Pretreatment with 100 mM glutathione (as described in Example 4)eliminated the formation (asterisk, p<0.05) of phagocytic (jellyfish)microglia following injury. This can be explained by the fact thatglutathione limits cellular damage and preserves the integrity of theglial limitans. Additionally, transcranial administration of glutathioneprevents the microglia from extending their processes and contacting theglial limitans following meningeal compression injury, as shown in FIG.10. Pretreatment with 100 mM glutathione (as described in Example 4)completely prevented this reaction.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1.-30. (canceled)
 31. A method of treating or preventing a disease ordisorder of the central nervous system (CNS) selected from the groupconsisting of brain injury, infection, degeneration of brain cells,brain edema, Alzheimer's disease, Parkinson's disease, and multiplesclerosis in a patient comprising administering transcranially aneffective amount of an anti-inflammatory agent to the patient, whereinthe anti-inflammatory agent is selected from the group consisting ofanti-oxidants, purinergic receptor inhibitors, and connexin hemichannelinhibitors.
 32. The method of claim 31, wherein the treating orpreventing comprises inhibiting, reducing, or eliminating (a) theformation of reactive microglia, (b) the recruitment of neutrophilsand/or monocytes, or (c) reducing the number of dead cells in the brainparenchyma or meninges in the patient.
 33. The method of claim 31,wherein the anti-inflammatory agent is an anti-oxidant selected from thegroup consisting of glutathione, ascorbic acid, lipoic acid, uric acid,carotenes, α-tocopherol, ubiquinols, and combinations thereof.
 34. Themethod of claim 33, wherein the anti-oxidant is glutathione.
 35. Themethod of claim 31, wherein the anti-inflammatory agent is a purinergicreceptor inhibitor.
 36. The method of claim 35, wherein the purinergicreceptor inhibitor is a P2X or P2Y receptor inhibitor.
 37. The method ofclaim 36, wherein the P2X or P2Y receptor inhibitor is a P2X₄ receptorinhibitor, a P2X₇ receptor inhibitor, a P2Y₆ receptor inhibitor, or aP2Y₁₂ inhibitor.
 38. The method of claim 37, wherein the P2X receptorinhibitor is a P2X₄ receptor inhibitor.
 39. The method of claim 38,wherein the P2X₄ receptor inhibitor is trinitrophenyl-ATP hydrate. 40.The method of claim 37, wherein the P2X inhibitor is a P2X₇ receptorinhibitor.
 41. The method of claim 40, wherein the P2X₇ receptorinhibitor is oxidized ATP.
 42. The method of claim 37, wherein the P2Yinhibitor is a P2Y₆ receptor inhibitor.
 43. The method of claim 42,wherein the P2Y₆ receptor inhibitor is N,N″-1,4-butanediylbis[N′-3-isothiocyanatophenyl)]thiourea (or MRS 2578).44. The method of claim 37, wherein the P2Y inhibitor is a P2Y₁₂receptor inhibitor.
 45. The method of claim 44, wherein the P2Y₁₂receptor inhibitor is MeSAMP.
 46. The method of claim 31, wherein theanti-inflammatory agent is an inhibitor of a connexin hemichannel. 47.The method of claim 16, wherein the connexin hemichannel inhibitor is(3β)-3-[(3-carboxypropanoyl)oxy]-11-oxoolean-12-en-30-oic acid (orCarbenoxolone).
 48. The method of claim 31, wherein theanti-inflammatory agent is administered via a transdermal patch.
 49. Themethod of claim 31, wherein the anti-inflammatory agent is administeredvia subcutaneous injection under the scalp.
 50. The method of claim 31,wherein the anti-inflammatory agent is administered via injection into acancellous bone of the skull bone.
 51. A kit comprising at least oneanti-inflammatory agent, optionally, a device for administering theanti-inflammatory agent to a head of a patient, and printed materialscontaining instructions for transcranially administering theanti-inflammatory agent to the patient having a disease or disorder ofthe central nervous system (CNS).